Two main aspects of chromosome structure and function are under investigation. The first is the development of techniques which allow the localization and identification of specific chromosome-associated proteins and nascent specific transcripts by electron microscopy of gently lysed nuclear contents prepared according to techniques developed by Miller. Ferritin-conjugated non-cross-reacting antibodies against histones H1 and H2B are interacted with chromatin in order to assess the distribution of each protein along individual chromatin strands. Antisera against the two unique large subunits of Xenopus laevis RNA polymerases are being generated. These will be used to locate both active and inactive enzymes along chromatin using polymethylmethacrylate spheres as the electron dense tag. This tagging procedure also is being applied to identification of specific hybrids of a purified cDNA with nascent transcripts in order to identify specific active genes. The other project involves purification of the centromere region of mouse metaphase chromosomes by selective DNase I digestion followed by characterization of the protein components using two-dimensional gels and the DNAs using density gradient centrifugation. These experiments are directed toward the elucidation of centromere-localized polypeptides and their putative interaction with specific DNA sequences of the centromere.